Galactooligosaccharides (GOS) are carbohydrates which are nondigestable in humans and animals comprising two or more galactose molecules, typically up to nine, linked by glycosidic bonds. GOS's may also include one or more glucose molecules. One of the beneficial effects of GOS's is their ability of acting as prebiotic compounds by selectively stimulating the proliferation of beneficial colonic microorganisms such as bacteria to give physiological benefits to the consumer. The established health effects have resulted in a growing interest in GOSs as food ingredients for various types of food.
The enzyme β-galactosidase (EC 3.2.1.23) usually hydrolyses lactose to the monosaccharides D-glucose and D-galactose. In the normal enzyme reaction of β-galactosidases, the enzyme hydrolyses lactose and transiently binds the galactose monosaccharide in a galactose-enzyme complex that transfers galactose to the hydroxyl group of water, resulting in the liberation of D-galactose and D-glucose. However, at high lactose concentrations some β-galactosidases are able to transfer galactose to the hydroxyl groups of D-galactose or D-glucose in a process called transgalactosylation whereby galacto-oligosaccharides are produced.
The genus Bífídobacterium is one of the most commonly used types of bacteria cultures in the dairy industry for fermenting a variety of diary products. Ingestion of Bifidobacterium-containing products furthermore has a health-promoting effect. This effect is not only achieved by a lowered pH of the intestinal contents but also by the ability of Bifidobacterium to repopulate the intestinal flora in individuals who have had their intestinal flora disturbed by for example intake of antibiotics. Bifidobacterium furthermore has the potential of outcompeting potential harmful intestinal micro-organisms.
Galacto-oligosaccharides are known to enhance the growth of Bifidobacterium. This effect is likely achieved through the unique ability of Bifidobacterium to exploit galacto-oligosaccharides as a carbon source. Dietary supplement of galacto-oligosaccharides is furthermore thought to have a number of long-term disease protecting effects. For example, galacto-oligosaccharide intake has been shown to be highly protective against development of colorectal cancer in rats. There is therefore a great interest in developing cheap and efficient methods for producing galacto-oligosaccharides for use in the industry for improving dietary supplements and dairy products.
A beta-galactosidase polypeptide from Ruminococcus/Blautia hansenii having 1807 amino acids (having SEQ ID NO: 12) is known from the database UniProt, 24 Nov. 2009, “Subname: Full=Beta-galactosidase” XP002591904 retrieved from EBI accession no. UNIPROT:C9LAL1.
A glycosidase having 1768 amino acids (having SEQ ID NO: 13) is known from the database UniProt, 14 Oct. 2008, “Subname: Full=Putative uncharacterised protein” XP002610554 retrieved from EBI accession no. UNIPROT:B5CQV4.
An extracellular lactase from Bifidobacterium bifidum DSM20215 truncated with approximately 580 amino acids (BIF3) has been described as a transgalactosylating enzyme in a solution containing lactose solubilised in water (Jorgensen et al. (2001), Appl. Microbiol. Biotechnol., 57: 647-652). In WO 2009/071539 a differently truncated fragment compared to BIF3 is described as resulting in efficient hydrolysis and very low production of GOS when tested in milk.
The Bifidobacterium bifidum lactase enzymes described above have the drawback of either requiring high lactose concentrations in order to exhibit transgalactosylase activity or pre-dominantly having beta-galactosylase (hydrolase) activity.
There is still a need to develop enzymes that are efficient at producing GOS and which furthermore can work at low lactose substrate levels such as in milk.